FRET Imaging of Rho GTPase Activity with Red Fluorescent Protein-Based FRET Pairs.
FRET Imaging of Rho GTPase Activity with Red Fluorescent Protein-Based FRET Pairs.
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使用基于红色荧光蛋白的 FRET 对对 Rho GTP 酶活性进行 FRET 成像。
DOI：
10.1007/978-1-0716-2035-9_2
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发表时间：
2022
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随着荧光蛋白 (FP) 和先进光学显微镜技术的发展，Förster 或荧光共振能量转移 (FRET) 已成为实时无创可视化各种生物过程（包括激酶活动）的强大工具，在活细胞和生物体中具有高时空分辨率。 FRET 可以在适当配置的显微镜中检测到荧光强度、寿命和各向异性的变化。在这里，我们描述了表达基于 FP 的 FRET 传感器的样品的制备，用于 RhoA 激酶、基于强度和寿命的 FRET 成像以及成像后数据分析。
With the development of fluorescent proteins (FPs) and advanced optical microscopy techniques, Förster or fluorescence resonance energy transfer (FRET) has become a powerful tool for real-time non-invasive visualization of a variety of biological processes, including kinase activities, with high spatiotemporal resolution in living cells and organisms. FRET can be detected in appropriately configured microscopes as changes in fluorescence intensity, lifetime, and anisotropy. Here we describe the preparation of samples expressing FP-based FRET sensors for RhoA kinase, intensity- and lifetime-based FRET imaging, and post-imaging data analysis.